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Biochemical Pharmacology (v.83, #9)
Histamine-gated ion channels in mammals? by Mark W. Fleck; Jeffrey L. Thomson; Lindsay B. Hough (pp. 1127-1135).
There is ample pharmacological and physiological evidence for yet unidentified histamine receptors in mammalian brain that are linked to a Cl− conductance. In invertebrates, two histamine-gated chloride channels (HisCl α1 and α2) are already well known. HisCl channels are members of the Cys-loop receptor superfamily of ligand-gated ion channels and are closely related to the mammalian GABAA and glycine receptors (GlyR). Indeed, they share particularly strong homology within the ligand binding and ion channel domains. Here we discuss the possibility that mammalian HisCl channels might exist among the known GABAA or GlyR subunits. Studies published to date support this hypothesis, including evidence for direct histamine gating of GABAA β homomers, histamine potentiation of GABAA αβ and αβγ heteromeric receptors, and GABAA receptor blockade by some antihistamines. We explore what is known about the binding-site structure, function and pharmacology of invertebrate HisCl channels and other histamine binding sites to support and inform a broader search for HisCl channels among the mammalian GABAA and GlyR subunits. The discovery and identification of HisCl-like channels in mammals would not only enhance understanding of inhibitory signaling and histamine function in the mammalian brain, but also provide new avenues for development of therapeutic compounds targeting this novel histamine site. This commentary is therefore intended to foster consideration of a novel and potentially important target of histamine and histaminergic drugs in the CNS.
Keywords: Histamine; HisCl channel; GABA receptor; Glycine receptor; Cimetidine; Binding site
The many faces of Janus kinase by Matthew M. Seavey; Pawel Dobrzanski (pp. 1136-1145).
Janus kinases have proved to be essential for many immunological processes but there is growing evidence that they also play a critical role in pathogenesis of many diseases including inflammatory diseases and cancer where they promote multiple steps of tumorigenesis. Several companies are in late stage clinical programs for the development of JAK kinase inhibitors and the first small molecule JAK inhibitor, Jakafi® (ruxolitinib) has been just approved for treatment of myeloproliferative neoplasms. Several other molecules are on the rise to treat arthritis, psoriasis and multiple types of cancer. This commentary will provide a review of the JAK kinase field as it pertains to small molecule inhibition for the treatment of cancer and autoimmune diseases with an emphasis on JAK2. The use of experimental and clinical inhibitors of JAK will be discussed for solid tumor and hematological malignancies, lupus, arthritis, colitis, neurological disorders, pain, diabetes and cardiovascular disease. In addition, it will review current paradigms in the field and treatment programs which could be complemented by small molecule inhibitors of Janus kinase.
Keywords: Janus kinase; Cancer; Autoimmunity; Small molecule; Inflammation
Sustained antitumor activity by co-targeting mTOR and the microtubule with temsirolimus/vinblastine combination in hepatocellular carcinoma by Qian Zhou; Vivian Wai Yan Lui; Cecilia Pik Yuk Lau; Suk Hang Cheng; Margaret Heung Ling Ng; Yijun Cai; Stephen Lam Chan; Winnie Yeo (pp. 1146-1158).
The mammalian target of rapamycin (mTOR) and the microtubules are prominent druggable targets for hepatocellular carcinoma (HCC). PI3K/Akt/mTOR activation is associated with resistance to microtubule inhibitors. Here, we hypothesized that co-targeting of mTOR (by mTOR inhibitor temsirolimus) and the microtubule (by microtubule-destabilizing agent vinblastine) would be more efficacious than single targeting in HCC models. In vitro studies showed that effective inhibition of mTOR signaling with temsirolimus alone was able to suppress HCC cell growth in a dose-dependent manner. Among five cell lines tested, Huh7 was the most temsirolimus-sensitive (IC50=1.27±0.06μM), while Hep3B was the most temsirolimus-resistant (IC50=52.95±17.14μM). We found that co-targeting of mTOR (by temsirolimus) and the microtubule (by vinblastine, at low nM) resulted in marked growth inhibition in Huh7 cells and synergistic growth inhibition in Hep3B cells (achieving maximal growth inhibition of 80–90%), demonstrating potent antitumor activity of this novel combination. In vivo studies showed that temsirolimus treatment alone for 1 week was able to inhibit the growth of Huh7 xenografts. Strikingly, the temsirolimus/vinblastine combination induced a significant and sustained antitumor activity (up to 27 days post-treatment), with effective reduction of tumor vessel density in both Huh7 and Hep3B xenograft models. Mechanistic investigation revealed that this marked antitumor effect was accompanied by specific and concerted down-regulation of several key anti-apoptotic/survival proteins (survivin, Bcl-2, and Mcl-1), which was not observed in single agent treatments. Our findings demonstrated that the potent anti-cancer activity of this co-targeting strategy was indeed mediated in parts by inhibition of these key survival/anti-apoptotic proteins.
Keywords: Abbreviations; HCC; hepatocellular carcinoma; mTOR; mammalian target of rapamycin; 4E-BP1; eIF4E binding protein; MVD; microvessel density; MDR; multi-drug resistant geneHepatocellular carcinoma; mTOR; Microtubule; Sustained antitumor activity; Anti-apoptotic/survival proteins
Vinca alkaloids cause aberrant ROS-mediated JNK activation, Mcl-1 downregulation, DNA damage, mitochondrial dysfunction, and apoptosis in lung adenocarcinoma cells by Wei-Hsin Chiu; Sheng-Jei Luo; Chia-Ling Chen; Jai-Hong Cheng; Chia-Yuan Hsieh; Chi-Yun Wang; Wei-Ching Huang; Wu-Chou Su; Chiou-Feng Lin (pp. 1159-1171).
Vinca alkaloids are clinically used to inhibit the growth of malignancy by interfering with microtubule polymerization. The purpose of this study was to identify the molecular mechanisms underlying growth inhibition as well as apoptosis in vinca alkaloid-treated lung adenocarcinoma cells. Consistent with nocodazole, treatment with vinorelbine (VNR) caused mitotic prometaphase arrest in a time-dependent manner, accompanied by cell apoptosis, dependent on both dose and time. VNR sequentially induced mitochondrial transmembrane potential (MTP) loss and caspase-dependent apoptosis following myeloid cell leukemia (Mcl) 1 downregulation. Prolonged activation of c-Jun N-terminal kinase (JNK) was required for vinca alkaloid- and nocodazole-induced apoptosis but not cell cycle arrest. Vinca alkaloids and nocodazole caused glutathione/reactive oxygen species (ROS) imbalance, and inhibiting ROS prevented prolonged JNK activation, decreased Mcl-1 levels, MTP loss, and apoptosis. Notably, cell size and granularity were enlarged in stimulated cells; unexpectedly, many ROS-producing mitochondria were accumulated followed by aberrant JNK-mediated mitochondrial dysfunction. Unlike cisplatin, which causes DNA damage in each phase of the cell cycle, VNR and nocodazole induced aberrant JNK-regulated DNA damage in prometaphase; however, inhibiting ATM (ataxia telangiectasia, mutated) and ATR (ATM and Rad3-related) did not reverse mitotic arrest or apoptosis. These results demonstrate an essential role of ROS in vinca alkaloid-induced aberrant JNK-mediated Mcl-1 downregulation and DNA damage followed by mitochondrial dysfunction-related apoptosis but not mitotic arrest.
Keywords: Vinca alkaloids; Lung adenocarcinoma; Mitotic arrest; Apoptosis; ROS; JNK; Mcl-1; Mitochondria; Caspase
Increased expression of VDAC1 sensitizes carcinoma cells to apoptosis induced by DNA cross-linking agents by Ossama Sharaf el dein; Cindy Gallerne; Catherine Brenner; Christophe Lemaire (pp. 1172-1182).
A major clinical problem regarding antitumoral treatment with DNA cross-linking agents such as cisplatin (Cisp), mechlorethamine (HN2) or its derivative melphalan (MLP) is intrinsic or acquired resistance to therapy, which frequently results from a resistance to apoptosis induction. In this study, aimed to identify novel sensitizing targets to DNA cross-linker-induced cell death, we demonstrated that MLP, Cisp and HN2 induce mitochondrial permeability transition pore (PTP)-mediated apoptosis in cervical and colon carcinoma cells. This apoptotic pathway is characterized by dissipation of the mitochondrial membrane potential, production of ROS, mitochondrial translocation of Bax, release of apoptogenic factors, caspase activation and nuclear alterations. The opening of PTP and subsequent apoptosis was reduced in Bax deficient cells and in cells with elevated Bcl-2 level, but not in cells invalidated for Bak. We further showed that, among the pro-apoptotic PTP regulators tested (VDAC1, creatine kinase, ANT1 and ANT3), exogenous overexpression of VDAC1 was the most effective in enhancing Cisp- and MLP-induced apoptosis. In addition, pharmacologically induced up-regulation of VDAC1 by the chemotherapeutic agent arsenic trioxide (As2O3) greatly sensitized HeLa cells to Cisp and MLP treatment. These data indicate that increased expression of VDAC1 appears as a promising strategy to improve DNA cross-linker-induced chemotherapy.
Keywords: Abbreviations; AIF; apoptosis-inducing factor; ANT; adenine nucleotide translocase; Ars; arsenic trioxide (As; 2; O; 3; ); Cisp; cisplatin; Δ; Ψ; m; mitochondrial transmembrane potential; HN2; mechlorethamine; IM; inner membrane; MLP; melphalan; MMP; mitochondrial membrane permeabilization; MPT; mitochondrial permeability transition; OM; outer membrane; PI; propidium iodide; PTP; permeability transition pore; ROS; reactive oxygen species; VDAC; voltage-dependent anion channelVDAC1; Permeability transition pore; Cisplatin; Melphalan; Apoptosis; Cancer
Inhibition of tumor cell growth, proliferation and migration by X-387, a novel active-site inhibitor of mTOR by Si-meng Chen; Jia-li Liu; Xiang Wang; Chris Liang; Jian Ding; Ling-hua Meng (pp. 1183-1194).
The mammalian target of rapamycin (mTOR), is deregulated in about 50% of human malignancies and exists in two complexes: mTORC1 and mTORC2. Rapalogs partially inhibit mTORC1 through allosteric binding to mTORC1 and their efficacy is modest as a cancer therapy. A few mTOR kinase inhibitors that inhibit both mTORC1 and mTORC2 have been reported to possess potent anticancer activities. Herein, we designed and synthesized a series of pyrazolopyrimidine derivatives targeting mTOR kinase domain and X-387 was identified as a promising lead. X-387 selectively inhibited mTOR in an ATP-competitive manner while sparing a panel of kinases from the PIKK family. X-387 blocked mTORC1 and mTORC2-mediacted signaling pathway in cell lines with activated mTOR signaling and in rapamycin-resistant cells. Specifically, X-387 inhibited phosphorylation of AKT at T308, which is thought to be a target of PDK1 but not mTOR. Such activity was not due to inhibition of PI3K since X-387 did not inhibit translocation of AKT to the cell membrane. X-387 induced autophagy as observed for other mTOR inhibitors, while induced autophagy is pro-survival since concurrent inhibition of autophagy by 3-MA reinforced the antiproliferative activity of mTOR inhibitors. X-387 also inhibited cell motility, which is associated with decrease in activity of small GTPases such as RhoA, Rac1 and Cdc42. Taken together, X-387 is a promising compound lead targeting mTOR and with a wide spectrum anticancer activity among tumor cell lines. The data also underscores the complexity of the mTOR signaling pathways which are far from being understood.
Keywords: X-387; mTOR kinase; Cancer therapy; Cell migration; Autophagy
Antiproliferative mechanisms of action of the flavin dehydrogenase inhibitors diphenylene iodonium and di-2-thienyliodonium based on molecular profiling of the NCI-60 human tumor cell panel by James H. Doroshow; Agnes Juhasz; Yun Ge; Susan Holbeck; Jiamo Lu; Smitha Antony; Yongzhong Wu; Guojian Jiang; Krishnendu Roy (pp. 1195-1207).
Flavoprotein-dependent reactive oxygen species (ROS) play a critical role in cytokine-mediated signal transduction in normal tissues and tumor cells. The flavoenzyme inhibitors diphenylene iodonium (DPI) and di-2-thienyliodonium (DTI) have been used to inhibit membrane-bound, flavoprotein-containing NADPH oxidases, including epithelial and leukocyte NADPH oxidases (Nox1-5 and Duox 1 and 2). Recent evidence suggests that DPI can decrease tumor cell proliferation; however, the molecular mechanisms involved remain poorly defined. To explore the mechanisms underlying DPI- and DTI-related tumor cell growth delay, we examined growth inhibition patterns produced by both agents in the NCI-60 tumor panel, and determined expression levels of Nox gene family members across these cell lines. Possible molecular targets were predicted using the COMPARE program. DPI was more potent than DTI (GI50: 10nM versus 10μM); DPI and DTI exposure produced unique patterns of growth inhibition when evaluated against the small molecule anticancer database of the National Cancer Institute. Growth inhibition profiling of DPI revealed a modest positive correlation with Nox1 levels; novel mechanisms of DPI and DTI action, including alterations in Stat, Erk1/2, and Akt pathways, were inferred by correlation with NCI-60 Affymetrix® array data. Exposure of HT-29 colon cancer cells, which express Nox1, to DPI and DTI confirmed their inhibitory effects on steady state ROS levels, and demonstrated decreased Stat, Erk1/2, and Akt signaling mediated by IL-4, IL-6, IL-13, and IL-22, possibly due to a concomitant increase in tumor cell phosphatase activity. These findings suggest that DPI and DTI may act therapeutically by altering ROS-related signal transduction.
Keywords: Abbreviations; ROS; reactive oxygen species; DPI; diphenyleneiodonium; DTI; di-2-thienyliodonium; NCI-60; National Cancer Institute panel of 60 human tumor cell lines; Nox; NADPH oxidase; Duox; dual oxidaseDiphenylene iodonium; Dithienyliodonium; Reactive oxygen; Stat signaling; NADPH oxidase; NCI-60 tumor panel
Anthracenedione–methionine conjugates are novel topoisomerase II-targeting anticancer agents with favorable drug resistance profiles by Chieh-Hua Lee; Mei-Yi Hsieh; Ling-Wei Hsin; Hsiang-Chin Chen; Su-Chi Lo; Jia-Rong Fan; Wan-Ru Chen; Hung-Wei Chen; Nei-Li Chan; Tsai-Kun Li (pp. 1208-1216).
Structure-associated drug resistance and DNA-unwinding abilities have greatly limited the clinical usage of anthracenediones, including mitoxantrone (MX) and ametantrone (AT), which intercalate into DNA and induce topoisomerase II (TOP2)-mediated DNA break. We studied a series of 1,4-bis(2-amino-ethylamino)MX- andAT-amino acidconjugates (M/AACs) and showed that abilities in cancer cell killing correlate with the amounts of chromosomal DNA breaks induced by M/AACs. Notably, the 1,4-bis-L/l-methionine-conjugated MAC (L/LMet-MAC) exhibits DNA-breaking, cancer cell-killing and anti-tumor activities rivaling those of MX. Interestingly,l- andd-form Met-M/AACs unwind DNA poorly compared to MX and AT. The roles of the two human TOP2 isozymes (hTOP2α and 2β) in the L/LMet-MAC-induced DNA breakage and cancer cell-killing were suggested by the following observations: (i) M/AAC-induced DNA breakage, cytotoxicity and apoptosis are greatly reduced in various TOP2-deficient conditions; (ii) DNA breaks induced by MACs are highly reversible and effectively antagonized by the TOP2 catalytic inhibitors; (iii) MACs induced differential TOP2-mediated DNA cleavage in vitro using recombinant hTOP2α proteins and the formation of hTOP2α/βcc in the cell culture system. Interestingly,d-aa-conjugated MACs often caused a lower level in hTOP2-mediated DNA breaks and cell-killing than the correspondingl-form ones indicating a steric-specific effect of MACs. Together, our results suggest that both enzyme- and DNA-drug interactions might contribute to TOP2-targeting by M/AACs. Furthermore, Met-MACs are poor substrates for the MDR1 transporter. Therefore, L/LMet-MAC represents a promising class of TOP2-targeting drugs with favorable drug resistance profiles.
Keywords: Abbreviations; MX; mitoxantrone; AT; ametantrone; MAC; mitoxantrone-amino acid conjugates; AAC; ametantrone-amino acid conjugates; MDR-1; multi-drug resistance 1; hTOP2; human topoisomerase II; TPT; topotecan; VP-16; etoposideTopoisomerase; Anthracenedione; Mitoxantrone; Cleavable complex; Multi-drug resistance
Acute sensitization of colon cancer cells to inflammatory cytokines by prophase arrest by Anton Kuratnik; Virginia E. Senapati; Rajeev Verma; Barbara G. Mellone; Anthony T. Vella; Charles Giardina (pp. 1217-1228).
Understanding how colon cancer cells survive within the inflammatory milieu of a tumor, and developing approaches that increase their sensitivity to inflammatory cytokines, may ultimately lead to novel approaches for colon cancer therapy and prevention. Analysis of a number of chemopreventive and therapeutic agents reveal that HDAC inhibitors are particularly adept at sensitizing colon cancer cells TNF or TRAIL mediated apoptosis. In vivo data are consistent with an interaction between SAHA and TNF in inducing apoptosis, as AOM-induced colon tumors express elevated levels of TNF and are more sensitive to SAHA administration. Cell cycle analysis and time-lapse imaging indicated a close correspondence between SAHA-induced prophase arrest and TNF or TRAIL-induced apoptosis. Prophase arrest induced by the Aurora kinase inhibitor VX680 likewise sensitized cells to TNF and TRAIL, with siRNA analysis pointing to Aurora kinase A (and not Aurora kinase B) as being the relevant target for this sensitization. We propose that agents that promote prophase arrest may help sensitize cancer cells to TNF and other inflammatory cytokines. We also discuss how circumvention of an early mitotic checkpoint may facilitate cancer cell survival in the inflammatory micro-environment of the tumor.
Keywords: Colon cancer; TNF; TRAIL; HDAC inhibitors; Aurora kinase inhibitors
Thiostrepton is an inducer of oxidative and proteotoxic stress that impairs viability of human melanoma cells but not primary melanocytes by Shuxi Qiao; Sarah D. Lamore; Christopher M. Cabello; Jessica L. Lesson; José L. Muñoz-Rodriguez; Georg T. Wondrak (pp. 1229-1240).
Pharmacological induction of oxidative and proteotoxic stress has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Guided by a differential phenotypic drug screen for novel lead compounds that selectively induce melanoma cell apoptosis without compromising viability of primary human melanocytes, we have focused on the cyclic pyridinyl-polythiazolyl peptide-antimicrobial thiostrepton. Using comparative gene expression-array analysis, the early cellular stress response induced by thiostrepton was examined in human A375 metastatic melanoma cells and primary melanocytes. Thiostrepton displayed selective antimelanoma activity causing early induction of proteotoxic stress with massive upregulation of heat shock ( HSPA6, HSPA1A, DNAJB4, HSPB1, HSPH1, HSPA1L, CRYAB, HSPA5, DNAJA1), oxidative stress ( HMOX1, GSR, SOD1), and ER stress response ( DDIT3) gene expression, confirmed by immunodetection (Hsp70, Hsp70B′, HO-1, phospho-eIF2α). Moreover, upregulation of p53, proapoptotic modulation of Bcl-2 family members (Bax, Noxa, Mcl-1, Bcl-2), and induction of apoptotic cell death were observed. Thiostrepton rapidly induced cellular oxidative stress followed by inactivation of chymotrypsin-like proteasomal activity and melanoma cell-directed accumulation of ubiquitinated proteins, not observed in melanocytes that were resistant to thiostrepton-induced apoptosis. Proteotoxic and apoptogenic effects were fully antagonized by antioxidant intervention. In RPMI 8226 multiple myeloma cells, known to be exquisitely sensitive to proteasome inhibition, early proteotoxic and apoptogenic effects of thiostrepton were confirmed by array analysis indicating pronounced upregulation of heat shock response gene expression. Our findings demonstrate that thiostrepton displays dual activity as a selective prooxidant and proteotoxic chemotherapeutic, suggesting feasibility of experimental intervention targeting metastatic melanoma and other malignancies including multiple myeloma.
Keywords: Abbreviations; AV; annexinV; BSO; l; -buthionine-S,R-sulfoximine; DCFH-DA; 2′,7′-dichlorodihydrofluorescein diacetate; DDIT3; DNA-damage-inducible transcript 3; EGR1; early growth response gene 1; FITC; fluorescein isothiocyanate; GSH; glutathione; HEMa; primary human epidermal melanocyte; Hsp; heat shock protein; HSPA1A; heat shock 70; kDa protein 1A; HSPA6; heat shock 70; kDa protein 6; HMOX1; heme oxygenase-1; NAC; N-acetyl-; l; -cysteine; PI; propidium iodide; ROS; reactive oxygen species; SDS-PAGE; sodium dodecylsulfate polyacrylamide gel electrophoresis; T; thiostrepton; Ub; ubiquitinMalignant melanoma; Oxidative and proteotoxic stress; Heat shock response; Proteasome; Thiostrepton
Processing of anthracycline-DNA adducts via DNA replication and interstrand crosslink repair pathways by R.A. Bilardi; K.-I. Kimura; D.R. Phillips; S.M. Cutts (pp. 1241-1250).
Anthracycline chemotherapeutics are well characterised as poisons of topoisomerase II, however many anthracyclines, including doxorubicin, are also capable of forming drug-DNA adducts. Anthracycline-DNA adducts present an unusual obstacle for cells as they are covalently attached to one DNA strand and stabilised by hydrogen bonding to the other strand. We now show that in cycling cells processing of anthracycline adducts through DNA replication appears dominant compared to processing via transcription-coupled pathways, and that the processing of these adducts into DNA breaks is independent of topoisomerase II. It has previously been shown that cells deficient in homologous recombination (HR) are hypersensitive to adduct forming treatments. Given that anthracycline-DNA adducts, whilst not true crosslinks, are associated with both DNA strands, the role of ICL repair pathways was investigated. Mus81 is a structure specific nuclease implicated in Holliday junction resolution and the resolution of branched DNA formed by stalled replication forks. We now show that ICL repair deficient cells (Mus81−/−) are hypersensitive to anthracycline-DNA adducts and ET-743, a compound which causes a chemically similar type of DNA damage. Further analysis of this mechanism showed that Mus81 does not appear to cause DNA breaks resulting from either anthracycline- or ET743-DNA adducts. This suggests Mus81 processes these novel forms of DNA damage in a fundamentally different way compared to the processing of classical covalent crosslinks. Improved understanding of the role of DNA repair in response to such adducts may lead to more effective chemotherapy for patients with BRCA1/2 mutations and other HR deficiencies.
Keywords: Anthracycline; DNA adduct; DNA repair; Homologous recombination; Interstrand crosslink
Arenobufagin, a bufadienolide compound from toad venom, inhibits VEGF-mediated angiogenesis through suppression of VEGFR-2 signaling pathway by Manmei Li; Shuai Wu; Zhong Liu; Wei Zhang; Jing Xu; Ying Wang; Junshan Liu; Dongmei Zhang; Haiyan Tian; Yaolan Li; Wencai Ye (pp. 1251-1260).
Angiogenesis is crucial for carcinogenesis and other angiogenic processes. Arenobufagin, one of the major components of toad venom, is a traditional Chinese medicine used for cancer therapy. It inhibits cell growth in several cancer cell lines. However, little is known about arenobufagin's anti-angiogenic activity. In this study, we showed that arenobufagin inhibited vascular endothelial growth factor (VEGF)-induced viability, migration, invasion and tube formation in human umbilical vein endothelial cells (HUVECs) in vitro. Arenobufagin also suppressed sprouting formation from VEGF-treated aortic rings in an ex vivo model. Furthermore, we found that arenobufagin blocked angiogenesis in a matrigel plugs assay. Computer simulations suggested that arenobufagin interacted with the ATP-binding sites of VEGFR-2 by docking. In addition, arenobufagin inhibited VEGF-induced VEGFR-2 auto-phosphorylation and suppressed the activity of VEGFR-2-mediated signaling cascades. Taken together, our findings demonstrate that arenobufagin is a specific inhibitor of VEGF-mediated angiogenesis.
Keywords: Abbreviations; HUVECs; human umbilical vein endothelial cells; VEGF; vascular endothelial growth factor; VEGFR-2; vascular endothelial growth factor receptor 2; RTK; receptor tyrosine kinase; DMSO; dimethyl sulphoxide; VEGFR-1; vascular endothelial growth factor receptor 1; bFGF; basic fibroblast growth factor; Erk1/2; extracellular signal-regulated kinase; EGF; epidermal growth factor; Hsp90; heat shock protein 90; FBS; fetal bovine serum; CCK-8; cell counting kit-8; H&E; hematoxylin–eosin; Co-IP; Co- immunoprecipitation; HIF-1α; hypoxia-inducible factor-1α; FAK; Focal adhesion kinaseArenobufagin; Angiogenesis; Vascular endothelial growth factor (VEGF); Vascular endothelial growth factor receptor 2 (VEGFR-2)
Role of endogenous hydrogen sulfide in neurogenic relaxation of rat corpus cavernosum by Mehdi Ghasemi; Ahmad R. Dehpour; Kevin P. Moore; Ali R. Mani (pp. 1261-1268).
Relaxation of corpus cavernosum during penile erection is mediated by a non-adrenergic non-cholinergic (NANC) neurotransmission and by the endothelium via the release of nitric oxide. Hydrogen sulfide (H2S) is an endogenous gaseous mediator which is a potent vasodilator and a neurotransmitter. This study was initiated to characterize the role of H2S in NANC neurogenic transmission in rat corpus cavernosum. The expression of H2S producing enzymes was assessed using RT-PCR as well as Western blotting and showed the expression of cystathionine γ-lyase (CSE) in rat corporal tissue. Homogenates from rat corpus cavernosum convertl-cysteine to H2S and this was partially inhibited by a CSE inhibitor, propargylglycine. Electrical stimulation of corporal tissue strips caused NANC relaxation. This neurogenic relaxation was significantly enhanced by inhibition of CSE by propargylglycine indicating that endogenously produced H2S may have a negative regulatory role in neurogenic relaxation of rat corpus cavernosum. To investigate this further we used physiologically relevant concentrations of exogenous NaHS, and showed that nanomolar concentrations could inhibit corporal relaxation induced by a nitroxyl (HNO) donor (Angeli's salt) but not with nitrosonium (NO+) or NO donors. This suggests that an interaction between endogenously produced H2S and nitroxyl (HNO) might be involved in erectile function.
Keywords: Corpus cavernosum; Cystathionine γ-lyase; Hydrogen sulfide (H; 2; S); Nitric oxide; Nitroxyl; Non-adrenergic non-cholinergic (NANC)
Chronic hydrogen-rich saline treatment attenuates vascular dysfunction in spontaneous hypertensive rats by Hao Zheng; Yong-Sheng Yu (pp. 1269-1277).
In hypertensive patients, increased oxidative stress is thought to be one important cause of vascular dysfunction. Recently, it has been suggested that hydrogen exerts a therapeutic antioxidant activity by selectively reducing hydroxyl radical and peroxynitrite, the most cytotoxic chemicals of reactive oxygen species (ROS). Herein, we investigated the protective effect of chronic treatment with hydrogen-rich saline (HRS) against vascular dysfunction in SHR and the underlying mechanism. The 8-week-old spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats (WKY) were randomized into HRS-treated (6ml/kg/day for 3 months, i.p.) and vehicle treated group. Treatment with HRS ameliorated vascular dysfunction including aortic hypertrophy and endothelial function in SHR. Treatment with HRS had no significant effect on blood pressure, but it significantly improved baroreflex function in SHR. Treatment with HRS abated oxidative stress, restored antioxidant enzymes including superoxide dismutase, glutathione peroxidase, and catalase, and suppressed NADPH oxidase. Furthermore, treatment with HRS depressed pro-inflammatory cytokines expression including IL-6 and IL-1β and suppressed NF-κB activation, restored mitochondrial function including ATP formation and membrane integrity. In addition, although treatment with HRS had no significant effect on nitric oxide amount in circulating or aorta, it suppressed endothelial nitric oxide synthase expression and upregulated dimethylarginine dimethylaminohydrolase 2 expression in SHR. In conclusion, treatment with HRS alleviates vascular dysfunction through abating oxidative stress, restoring baroreflex function, suppressing inflammation, preserving mitochondrial function, and enhancing nitric oxide bioavailability.
Keywords: Abbreviations; HRS; hydrogen-rich saline; NF-κB; NF-kappa B; DDAH2; dimethylarginine dimethylaminohydrolase 2; SOD; superoxide dismutase; eNOS; endothelial nitric oxide synthase; GPx; glutathione peroxidase; ROS; reactive oxygen species; SBP; systolic blood pressure; DBP; diastolic blood pressure; HP; heart period; SBPV; systolic blood pressure variability; DBPV; diastolic blood pressure variability; BRS; baroreflex sensitivity; MDA; malondialdehyde; O; 2; −; superoxide; OONO; −; peroxynitrite; AW; aortic weight; WT; wall thickness; SHR; spontaneously hypertensive rats; WKY; Wistar-Kyoto ratsHydrogen-rich saline; Spontaneously hypertensive rat; Oxidative stress; Vascular dysfunction; Hypertension
Dihydroartemisinin exhibits antitumor activity toward hepatocellular carcinoma in vitro and in vivo by Chris Zhiyi Zhang; Haitao Zhang; Jingping Yun; George Gong Chen; Paul Bo San Lai (pp. 1278-1289).
Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua L., has been shown to exhibit inhibitory effects on human cancer cells. However, its antitumor ability toward hepatocellular carcinoma (HCC) has not been studied. In this study, we demonstrated that DHA significantly inhibited HCC cell growth in vitro and in vivo via inducing G2/M cell cycle arrest and apoptosis. The induction of p21 and the inhibition of cyclin B and CDC25C contributed to DHA-induced G2/M arrest. DHA-induced apoptosis was associated with mitochondrial membrane depolarization, release of cytochrome c, activation of caspases, and DNA fragmentation. Activation of caspase 9 and caspase 3, but not caspase 8, was detected in DHA-treated cells. Attenuation of apoptosis in cells pretreated with Z-VAD-FMK suggested the involvement of caspase cascade. Furthermore, p53 facilitated apoptosis caused by DHA. Bcl-2 family proteins were also responsible for DHA-induced apoptosis. DHA exposure decreased Mcl-1 expression but increased the levels of Noxa and active Bak. Bak was released from the Mcl-1/Bak complex due to the decline of Mcl-1. Further study revealed that Mcl-1 was rapidly degraded in DHA-treated cells and that DHA-induced apoptosis was largely inhibited by overexpression of Mcl-1 or RNAi-mediated decrease of Bak and Noxa. In a HCC-xenograft mouse model, the intraperitoneal injection of DHA resulted in significant inhibition of HCC xenograft tumors. Taken together, our data, for the first time, demonstrate the potential antitumor activity of DHA in HCC.
Keywords: Dihydroartemisinin; Apoptosis; Mcl-1; Bak; Hepatocellular carcinoma
Arsenic mobilizes Langerhans cell migration and induces Th1 response in epicutaneous protein sensitization via CCL21: A plausible cause of decreased Langerhans cells in arsenic-induced intraepithelial carcinoma by Chih-Hung Lee; Chien-Hui Hong; Chia-Li Yu; Li-Fang Wang; Björn E. Clausen; Wei-Ting Liao; Shau-Ku Huang; Gwo-Shing Chen; Hsin-Su Yu (pp. 1290-1299).
Arsenic, still a significant environmental threat in several regions in the world, induces various cancers, including lungs, skin, and bladder. Arsenic-induced Bowen's disease (As-BD) is generally an indolent cutaneous intraepithelial carcinoma in susceptible people. Patients with As-BD have been found to have attenuated contact hypersensitivity. Skin samples collected from these patients have reduced numbers of Langerhans cells (LCs), the major epidermal antigen presenting cells expressing Langerins. This study uses an epicutaneous protein sensitization model to investigate the mechanism through which LCs are decreased in As-BD. It further investigates the possibility that arsenic alters LC migration and polarizes Th responses. To do this, we patch-sensitized Balb/c mice or DT-treated Langerin-DTR mice (conditional depletion of Langerin+ cells) with OVA or PBS, and fed them water containing 300ppb arsenic or regular water for 200μl for five days. Ninety-six hours after OVA sensitization, Langerin+EpCAM+ cells in arsenic-treated WT mice were significantly increased in draining lymph nodes and decreased in epidermis without changes in the dermis. Lymph node cells from arsenic-treated WT mice were found to proliferate more than lymph node cells from control PBS-treated mice after OVA challenge in vitro. They also secreted more IFN-γ and IL-12, but not IL-4, IL-13, or IL-17. However, cell proliferation and the induction of IFN-γ by arsenic were found to be abolished in DT-treated Langerin-DTR mice. The expressions of CCL21 and CXCL12 were also increased in lymph nodes from arsenic-treated WT mice. The administration of a neutralizing antibody against CCL21, but not CXCL12, abolished the increase of LCs in lymph nodes in vivo. The results of this study, the first to study oral arsenic polarization of Th1 responses in epicutaneous protein sensitization through CCL21-mediated LC migration, suggest the chronicity of As-BD without invasion might result from enhanced Th1 responses and altered LC migrations by arsenic.
Keywords: Abbreviations; As-BD; arsenic-induced Bowen's disease; DTH; delayed-type hypersensitivity; LNs; lymph nodes; DNFB; 2,4-dinitrofluorobenzene; LCs; Langerhans cellsArsenic; Langerhans cells; IFN-gamma; CCL21; Bowen's disease
Additive activation of glucokinase by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and the chemical activator LY2121260 by Simone Baltrusch; Heike Schmitt; Anke Brix; Sara Langer; Sigurd Lenzen (pp. 1300-1306).
The glucose phosphorylating enzyme glucokinase plays a crucial role in stimulus-secretion coupling in pancreatic beta cells and in glucose metabolism in liver. Glucose mediates a shift of the enzyme's conformational equilibrium towards the closed conformation with high glucokinase activity. Further activation of glucokinase is endogenously mediated by interaction with the bisphosphatase domain (FBPase-2) of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) and can be achieved also by a new class of glucokinase activators (GKA), chemical compounds that might be suited for type 2 diabetes therapy. While FBPase-2 increased only the phosphorylating capacity of glucokinase, the GKA LY2121260 augmented in addition the affinity of glucokinase for glucose. PFK-2/FBPase-2 but not LY2121260 antagonized glucokinase inhibition by the competitive glucokinase inhibitor mannoheptulose at increasing glucose concentrations. Interestingly, an additive activation of glucokinase was observed by use of recombinant FBPase-2 together with LY2121260. This new crucial observation could be confirmed with cellular extracts containing the glucokinase and PFK-2/FBPase-2 proteins. Addition of LY2121260 resulted in a further significant increase in glucokinase activity. Because the glucokinase-PFK-2/FBPase-2 complex was conserved under LY2121260 treatment as shown by size exclusion chromatography a concerted action of both activators towards the closed active glucokinase conformation can be anticipated. Thus, as a result of the additive effect of both activators on glucokinase activity, the largest increase of glucose-induced insulin secretion was observed in the combined presence of PFK-2/FBPase-2 and LY2121260.
Keywords: Abbreviations; GK; glucokinase; PFK-2/FBPase-2; 6-phosphofructo-2-kinase/fructose-2,6-bisphosphataseGlucokinase; 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Glucokinase activator LY2121260; Pancreatic beta-cell; Insulin secretion
Purification, molecular cloning and functional characterization of HelaTx1 ( Heterometrus laoticus): The first member of a new κ-KTX subfamily by Thomas Vandendriessche; Ivan Kopljar; David Paul Jenkins; Elia Diego-Garcia; Yousra Abdel-Mottaleb; Elke Vermassen; Elke Clynen; Liliane Schoofs; Heike Wulff; Dirk Snyders; Jan Tytgat (pp. 1307-1317).
Given their medical importance, most attention has been paid toward the venom composition of scorpions of the Buthidae family. Nevertheless, research has shown that the venom of scorpions of other families is also a remarkable source of unique peptidyl toxins. The κ-KTx family of voltage-gated potassium channel (VGPC) scorpion toxins is hereof an example. From the telson of the scorpion Heterometrus laoticus (Scorpionidae), a peptide, HelaTx1, with unique primary sequence was purified through HPLC and sequenced by Edman degradation. Based on the amino acid sequence, the peptide could be cloned and the cDNA sequence revealed. HelaTx1 was chemically synthesized and functionally characterized on VGPCs of the S haker-related, Shab-related, Shaw-related and Shal-related subfamilies. Furthermore, the toxin was also tested on small- and intermediate conductance Ca2+-activated K+ channels. From the channels studied, Kv1.1 and Kv1.6 were found to be the most sensitive (Kv1.1 EC50=9.9±1.6μM). The toxin did not alter the activation of the channels. Competition experiments with TEA showed that the toxin is a pore blocker. Mutational studies showed that the residues E353 and Y379 in the pore of Kv1.1 act as major interaction points for binding of the toxin. Given the amino acid sequence, the predicted secondary structure and the biological activity on VGPCs, HelaTx1 should be included in the κ-KTX family. Based on a phylogenetic study, we rearranged this family of VGPC toxins into five subfamilies and suggest that HelaTx1 is the first member of the new κ-KTx5 subfamily.
Keywords: Scorpion toxin; Voltage-gated potassium channel; Electrophysiology; Patch clamp; cDNA; Heterometrus laoticus